I think to change from .NET 3.5 to 4 will result in some down time unless you make a copy of the site at a different hostname and then change the dns of the main hostname after the site is working.
500 is the status code of all web requests that result in an error, need to know the detail of the error to resolve it.
When changing like this errors are going to happen because the .NET 4 package files will cause an error if the application pool is 3.5 and the 3.5 package of mojoPortal will also result in an error if the application pool is for 4. There are incompatible difference in Web.config between the different frameworks. It is technically possible to run the mojo 3.5 package under .NET 4 with web.config modifications because older .NET assemblies can run in the newer framework but the mojo package for .NET 4 will never load under .NET 3.5 and the first problem will be web.config. These web.config errors are discussed in the 9th and 10th items in our faq.
Web.config errors means it fails to load the web.config so no settings from web.config are applied since it failed to load and the site will not load. In this case the error may not be in the mojoportal log but the detail will be in the windows application log in event viewer on the server.
To change to .NET 4 you would prepare the files on your local machine, transfer machinekey from old web.config to new one, upload all the files and of course errors will happen until all the files are uploaded and the application pool is changed to 4.
Once that is done if you still get an error we still need the detail of the error one way or another, either form the mojoportal log or from the windows application log in event viewer which your host could access for you. Once the error detail is known the solution will be clear. Probably some file failed to upload or something.
Note also that if you have any add on products you will need to update to .NET 4 version of those too as part of preparing the package on your local machine before uploading is the ideal time to update those files.
Hope that helps,